The central objective of this project is to elucidate the roles of epigenetics and chromatin states in RNA splicing regulation. Eukaryotic cells generate astonishing regulatory diversity and as a consequence exceedingly complex phenotypes from a finite set of genes. Alternative pre-mRNA splicing plays an essential role in creating this regulatory diversity by generating multiple RNA isoforms from a single gene. Traditionally, splicing was considered as a post-transcriptional process, and studies of splicing regulation have largely focused on the roles of cis splicing regulatory elements and their interactions with canonical RNA-binding splicing factors. However, recent studies of eukaryotic epigenomes and transcriptomes have revealed a surprisingly complex picture of splicing regulation shaped by chromatin states and epigenetic marks. Exons are characterized by increased levels of nucleosome positioning, DNA methylation, and certain histone modifications. Many introns are spliced co-transcriptionally when the nascent RNAs are tethered to the chromatin, and changes in the transcription elongation rate or epigenetic marks can influence exon splicing patterns. Despite these exciting findings, many questions about epigenetic regulation of splicing remain unresolved. We propose to systematically investigate chromatin and epigenetic regulation of RNA splicing, by taking advantage of the broad and deep epigenome and transcriptome data generated by the Epigenome Roadmap project. By correlating transcriptome profiles to epigenome profiles across diverse cell types, we aim to address a series of important questions regarding epigenetic regulation of co-transcriptional and post-transcriptional RNA splicing. In three aims, we will investigate epigenome-splicing correlation in diverse tissues and cell types (Aim 1), identify combinatorial chromatin states and long-range interactions associated with splicing (Aim 2), and elucidate how epigenetic determinants affect chromatin-associated splicing (Aim 3). The proposed studies will significantly advance our understanding of splicing regulation, and how epigenetic signals influence alternative splicing in normal and diseased cells. In addition, through the proposed work we will develop novel computational methods for linking epigenome signatures to RNA splicing patterns. We anticipate that these tools will be of broad interest and utility to researchers studying epigenome and transcriptome regulation in diverse biological systems.